In many cases malignant transformation can be directly linked to activation of the STAT family of transcription factors. Two major STAT isoforms have been characterized: the full-length transcription factor and C-terminal truncated isoforms that lack the transactivation domain. Constitutive activation of STAT5 proteins has been demonstrated in many diverse human cancer cell lines and clinical tumors including those of the prostate. Furthermore, our studies provide evidence that STAT5B, but not STAT5A, contributes to tumor progression. Increased activation of STAT5 correlates with aggressive behavior of prostate cancer while the naturally occurring dominant-negative truncated isoform, STAT5?B blocks tumorigenic abilities cancer cells. Therefore, the overall hypothesis of this proposal is that ectopic expression of the dominant-negative truncated isoform STAT5?B, a tumor suppressor, in place of the proto-oncogene STAT5B reverses carcinogenesis. The rationale of this study is that validation of this hypothesis can lead to novel approaches and the development of disease-intervention strategies in the treatment of prostate cancer and likely, other malignances. We propose to substitute expression of STAT5B (proto-oncogene) with the dominant-negative truncated isoform STAT5?B (tumor suppressor) applying pHLIP nanotechnology for delivery of splice-switching oligonucleotides to block alternative splicing. The proposed work combines recently acquired knowledge on regulation and action of specific genes involved in prostate cancer progression with a novel nanotechnology-based approach for targeted delivery of DNA specifically in tumors. Aim1: To investigate the efficacy of the steric-blocking by splice-switching oligonucleotide (SSO) conjugates with pH insertion peptide (pHLIP) to block alternative splicing of STATs mRNA in vitro. The hypothesis of this aim is that pHLIP is well suited to target delivery of splice switching DNA into tumor cells. The rationale of this aim is that optimization of SSO delivery employing pHLIP is necessary to attempt this strategy in vivo. Aim2: To test the ability of pHLIP-SSOs directed to induce expression of STAT5?B to block prostate cancer in vivo. The hypothesis of this aim is that application of the pHLIP-SSO technology will induce ectopic expression of STAT5?B (tumor suppressor) in place of STAT5B (proto-oncogene) in the Athymic Nude Mouse Model System. The rationale of this aim is based on the overall hypothesis of this proposal that ectopic expression of the dominant-negative truncated isoform STAT5?B in place of the proto-oncogene STAT5B can reverse carcinogenesis. The modulation of STAT5B isoform expression represents a model for the development of a novel therapeutic approach not only for prostate cancer but also for other diseases caused by defective pre-mRNA splicing. The successful completion of this work is anticipated to open new avenues for chemotherapeutic disease intervention strategies based on the proposed combination of pHLIP nanotechnology and a novel approach to switching expression from a proto- oncogene to a tumor suppressor. RELEVANCE TO PUBLIC HEALTH: This project aims to develop an innovative strategy for therapeutic approaches not only for prostate cancer but also for other diseases caused by defective pre-mRNA splicing. Completion of this proposal may open new avenues for chemotherapeutic disease intervention strategies based on the proposed combination of pHLIP nanotechnology and a novel approach to switching expression from a proto-oncogene to a tumor suppressor.